The present invention relates to the field of genetic engineering, particularly the field of gene therapy.
Gene therapy attempts to treat diseases caused by congenital or acquired genetic defects, namely gene disorders, by substituting or supplementing defective genes with normal genes. Although various treatment methods for gene therapy have been investigated, only a very limited number of the methods to date have met with success, including the treatment of adenosine deaminase (ADA) deficiency. This is mainly because the methods for efficiently introducing a therapeutic gene into target cells and the methods for expressing an introduced gene in the cell have not yet been established. So far, liposomes, HVJ-liposomes, retroviruses, and the like have been employed as carriers introducing the therapeutic gene into target cells. However, none of them are satisfactory in gene introduction efficiency. Various attempts have been made to increase the expression efficiency of the introduced gene, by, for example, improving the promoter. However, in each case the expression efficiency of the desired gene was still poor, and the quantity of the gene product was insufficient to afford gene therapy. Thus, in the field of gene therapy, a vector that enables a high level expression of a therapeutic gene in a variety of target cells has been sought.
In the field of hematology, Tec tyrosine kinase, a protein thought to participate in the proliferation of hematopoietic stem cells, is highly expressed in mouse liver, and is also expressed in the kidney, heart, and ovary (Oncogene, 5, 1781-1786 (1990)). In humans, Tec tyrosine kinase is highly expressed in a wide range of blood and lymphoid cells (LEUKEMIA, 8, 1663-1672 (1994)). However, human Tec promoter has not been reported so far, and its function has only been predicted from the study on the corresponding mouse sequence.
An objective of the present invention is to provide a vector having incorporated within it a promoter functioning efficiently in a wide variety of blood and lymphoid cells, and the cells of organs such as the liver, thereby providing a gene therapy technique targeting blood and lymphoid cells.
The present inventors noted that Tec tyrosine kinase, a protein thought to participate in the proliferation of hematopoietic stem cells, is highly expressed in a wide variety of blood cells, lymphoid cells, and the cells of organs such as the liver. The inventors isolated the promoter of Tectyrosine kinase from a mouse genomic DNA, constructed a vector with the promoter incorporated within it ligated an exogenous gene adjacently downstream of it, and attempted to express the exogenous gene in the cells. As a result, they found that the exogenous gene was actually expressed in the cells at a high level. However, in view of species specificity and safety, human Tec promoter appears to be more useful than mouse Tec promoter when used in gene therapy for treating humans. Thus, a human genomic library was screened for Tec promoter, and a DNA fragment of 3,129 bp in length was isolated and sequenced (FIG. 3). Thus, the present invention relates to:
(1) a DNA comprising the nucleotide sequence of SEQ ID NO:10 and having promoter activity;
(2) an expression vector comprising the DNA of (1); and
(3) a cell carrying the expression vector of (2).
In the present invention, xe2x80x9ca DNA having promoter activityxe2x80x9d means a DNA having activity to induce the transcription of a DNA region adjacent to the DNA. The present invention includes a DNA containing a part or whole of the nucleotide sequence of SEQ ID NO:1, and having promoter activity as well as the DNA having the nucleotide sequence of SEQ ID NO:1. Preferably, the DNA of the present invention has a length of at least about 50 bp, more preferably at least about 100 bp, even more preferably at least about 200 bp, and most preferably at least about 300 bp. The present invention also includes a DNA containing a part or whole of the nucleotide sequence of SEQ ID NO: 9, and having promoter activity as well as the DNA having the nucleotide sequence of SEQ ID NO: 9. Preferably, the DNA of the present invention has a length of at least about 50 bp, preferably at least about 100 bp, more preferably at least about 150 bp, even more preferably at least about 200 bp, and most preferably at least about 300 bp, for example, at least about 400 bp or at least about 500 bp. The present invention also relates to at least 1 kb sequence located in between approximately 1 kb to 2 kb upstream from the 3xe2x80x2 terminus of the sequence of SEQ ID NO: 9. The inventors have studied the promoter activity of this sequence, using a reporter assay in which luciferase luminescence is measured.
According to the present invention, any vector for use in gene introduction can basically be used as a xe2x80x9cvectorxe2x80x9d into which the DNA having promoter activity is to be introduced. Particularly in gene therapy, viral vectors, such as retrovirus vectors, adenovirus vectors, or adeno associated virus vectors, and non-viral vectors such as liposomes should be used.
Any cell is included in the xe2x80x9ccell carrying the expression vectorxe2x80x9d of the present invention. Cells that have been confirmed to actually express the Tec tyrosine kinase gene, including blood cells and lymphoid cells, such as hematopoietic stem cells, myeloid cells, B cells, and T cells, and the cells of internal organs such as the liver, kidney, heart, and ovary should be used.
One skilled in the art may be able to prepare and identify a DNA containing a part of the nucleotide sequence of SEQ ID NO: 9, and having promoter activity by digestion with exonucleases such as ExoIII and Bal31 or restriction enzymes and by examining the promoter activity of the DNA as follows. The DNA is ligated upstream of a reporter gene, such as luciferase gene, on an expression vector containing no promoter, for example, pUC00Luc, etc. By introducing the vector into cells highly expressing the Tec mRNA such as BA/F3 cells, test cells for assays of reporter gene activity are prepared. After cultured, the cells are harvested and subjected to the assays. For example, the assays for luciferase can be performed according to the ordinary method (the method according to the manual of xe2x80x9cLuciferase Assay Systemxe2x80x9d (Promega)). As a control, cells into which the same expression vector containing no promoter as mentioned above has been introduced are used without ligating any DNA upstream of the reporter gene. If the expression level of the reporter gene is significantly higher in the test cells than in the control cells, it is confirmed that the DNA has promoter activity.